Treatment of viral diseases with recombinant interferon α

ABSTRACT

This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. The super-compound interferon possesses anti-viral or anti-tumor activity and, therefore, is useful in preventing and treating viral diseases and cancers. This invention also provides an artificial gene which codes for the super-compound interferon or its equivalent. Finally, this invention provides methods for producing recombinant super-compound interferon or its equivalent and various uses of said interferon.

The application disclosed herein is a continuation of U.S. Ser. No. 10/928,956, filed Aug. 26, 2004 which claims priority of U.S. Ser. No. 60/498,449, Filed Aug. 28, 2003. This application claims priority of Indian Application No. 279/MUM/2004, filed Mar. 5, 2004, and Indian Application No. 280/MUM/2004, filed Mar. 5, 2004. The contents of the preceding applications are hereby incorporated in their entities by reference into this application.

Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention is related to a recombinant super-compound interferon (rSIFN-co) with changed spatial configuration. One characteristic of rSIFN-co in this invention is that it inhibits DNA (deoxyribonucleic acid) duplication of the hepatitis B virus as well as the secretion of HBsAg and HBeAg.

BACKGROUND OF THE INVENTION

rSIFN-co is a new interferon molecule constructed with the most popular conservative amino acid found in natural human α-IFN subtypes using genetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 have described it. rSIFN-co had been proved to have broad-spectrum IFN activity and virus- and tumor-inhibition and natural killer cell activity. U.S. Pat. No. 5,372,808 by Amgen, Inc. addresses treatment rSIFN-co. Chinese Patent No. 97193506.8 by Amgen, Inc. addresses re-treatment of rSIFN-co on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment of rSIFN-co on hepatitis B and hepatitis C.

The United States Food and Drug Administration (FDA) authorized Amgen, Inc. to produce rSIFN-co with E. Coli. for clinical hepatitis C treatment at the end of 1997.

Hepatitis B patients can be identified when detecting HBsAg and HBeAg. α-IFN is commonly used in clinics to treat hepatitis B. IFN binds superficial cell membrane receptors, inhibiting DNA and RNA (ribonucleic acid) duplication, including inducing some enzymes to prevent duplication of the virus in hepatitis-infected cells. All IFNs can inhibit only the DNA duplication of viruses, not the e and s antigen.

This disclosure describes recombinant super-compound interferon, method to produce the same, and uses thereof.

SUMMARY OF THE INVENTION

This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. An equivalent is a molecule which is similar in function to the super-compound interferon. The super-compound interferon possesses anti-viral or anti-tumor activity. This invention also provides artificial gene which codes for the super-compound interferon or its equivalent.

This invention provides a process for production of recombinant super-compound interferon comprising introducing an artificial gene with selected codon preference into an appropriate host, culturing said introduced host in an appropriate condition permitting expression of said super-compound interferon, and harvesting the expressed super-compound interferon.

This invention provides a composition comprising the recombinant super-compound interferon or its equivalent and a suitable carrier. This invention further provides a pharmaceutical composition comprising the recombinant super-compound interferon or its equivalent and a pharmaceutically acceptable carrier.

This invention provides a method for treating viral diseases or tumor in a subject comprising administering to the subject an effective amount of the super-compound interferon or its equivalent.

This invention provides the above-described method wherein super-compound interferon was administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via an inspirator.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. rSIFN-co cDNA sequence designed according to E. Coli. codon usage and deduced rSIFN-co amino acid sequence

FIG. 2. Sequence of another super-compound interferon

FIG. 3. Diagram of pLac T7 cloning vector plasmid

FIG. 4. Diagram of pHY-4 expression vector plasmid

FIG. 5. Construction process of expression plasmid pHY-5

FIG. 6-A. Circular Dichroism spectrum of Infergen®

(Tested by Analysis and Measurement Center of Sichuan University)

Spectrum range: 250 nm-190 nm

Sensitivity: 2 m°/cm

Light path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml IFN-con1, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na₂PO₄, pH7.0.

Infergen® (interferon alfacon-1), made by Amgen Inc., also known as consensus interferon, is marketed for the treatment of adults with chronic hepatitis C virus (HCV) infections. It is currently the only FDA approved, bio-optimized interferon developed through rational drug design and the only interferon with data in the label specifically for non-responding or refractory patients. InterMune's sales force re-launched Infergen® in Jan. 2002 with an active campaign to educate U.S. hepatologists about the safe and appropriate use of Infergen®, which represents new hope for the more than 50 percent of HCV patients who fail other currently available therapies.

FIG. 6-B. Circular Dichroism spectrum of Infergen® From Reference [Journal of Interferon and Cytokine Research. 16:489-499(1996)]

Circular dichroism spectra of concensus interferon subforms. Concensus interferon was fractionated using an anion exchange column. Samples were dialyzed into 10 mM sodium phosphate, pH 7.4. Measurements were made on Jasco J-170 spectopolarimeter, in a cell thermostat at 15° C. (-), acylated form; (--) cis terminal form; ( . . . ), met terminal form. A. Far UV Spectrum. B. Near UV Spectrum.

FIG. 6-C. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 320 nm-250 nm

Sensitivity: 2 m°/cm

Light path: 2 cm

Equipment: Circular Dichroism J-500C

Samples: contains 0.5 mg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na₂PO₄, pH7.0.

FIG. 6-D. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 250 nm-190 nm

Sensitivity: 2 m°/cm

Light path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na₂PO₄, pH7.0.

Clearly, as evidenced by the above spectra, the secondary or even tertiary structure of rSIFN-co is different from Infergen®.

FIG. 7. Comparison of Inhibition Effects of Different Interferons on HBV Gene Expression

FIG. 8A. Curves of Changes of Body Temperature in Group A (5 patients)

This figure is the record of body temperature changes of 5 patients in Group A.

FIG. 8B. Curves of Changes of Body Temperature in Group A (6 patients)

This figure is the record of body temperature changes of the other 6 patients in Group A.

FIG. 8C. Curves of Changes of Body Temperature in Group B (5 patients)

This figure is the record of body temperature changes of 5 patients in Group B.

FIG. 8D. Curves of Changes of Body Temperature in Group B (5 patients)

This figure is the record of body temperature changes of the other 5 patients in Group B.

FIG. 9. rsIFN-co Crystal I

FIG. 10. rsIFN-co Crystal II

FIG. 11. The X-ray Diffraction of rsIFN-co Crystal

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for producing a recombinant super-compound interferon with changed spatial configuration and enhanced antiviral activity comprising steps of:

-   -   (a) Introducing nucleic acid molecule which codes for said         interferon with preferred codons for expression to an         appropriate host; and     -   (b) Placing the introduced host in conditions allowing         expression of said interferon.

This invention provides the method for producing interferon, further comprising recovery of the expressed interferon.

This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. This invention reveals that proteins with the same primary sequence might have different biological activities. As illustrated in the following example, this invention discloses two proteins with identical amino acid sequences but with different activities. The efficacy of this activity may sometimes be improved and, sometimes, the protein with changed spatial configuration would reveal new function.

An equivalent is a molecule which is similar in function to the compound interferon. An equivalent could be a deletion, substitution, or replacement mutant of the original sequence. Alternatively, it is also the intention of this invention to cover mimics of the recombinant super-compound interferon. Mimics could be a peptide, polypeptide or a small chemical entity.

The interferon described herein includes but is not limited to interferon α, β, or ω. In an embodiment, it is IFN-1a, IFN-2b or other mutants.

In an embodiment, the super-compound interferon disclosed has higher efficacy than the interferon described in U.S. Pat. Nos. 4,695,623 or 4,897,471. This super-compound interferon is believed to have unique secondary or tertiary structure. (See e.g. FIG. 6.)

The super-compound interferon described herein has spatial structure change(s) resulting from the changes of its production process.

The above-described super-compound interferon may be produced by a high-efficiency expression system which uses a special promoter. In an embodiment, the promoter is P_(BAD). As could be easily appreciated by other ordinary skilled artisans. Other inducible promoters, such as heat shock promoters or heavy metal inducible promoters, may be used in this invention.

The super-compound interferon may also be produced with its gene as artificially synthesized cDNA with adjustment of its sequence from the wild-type according to codon preference of E. Coli. Extensive discussion of said codon usage (preference) may be found in U.S. Pat. No. 4,695,623. See e.g. column 6, line 41-column 7, line 35.

The above-described super-compound interferon possesses anti-viral or anti-tumor activity, and; therefore, is useful in inhibiting, preventing and treating viral diseases, tumors, or cancers.

As used herein, viral diseases include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections caused by Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, other herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia virus I, human T-cell leukemia virus II, or human T-cell leukemia virus III.

Viral upper respiratory infection, alternative names common cold, colds. This is a contagious viral infection of the upper respiratory tract characterized by inflammation of the mucous membranes, sneezing, and a sore throat. It is usually caused by over 200 different viruses, known as rhinoviruses. Colds are not caused by the same viruses responsible for influenza. Colds are spread through droplets from the coughing or sneezing of others with a cold or by hand contact with objects contaminated by someone with a cold. The incidence of colds is highest among children, and the incidence decreases with age because immunity to the virus causing the cold occurs after the illness. Gradually, immunity to a wide variety of viruses that cause colds is developed in adults. Children may have 10 colds a year, and adults may have 3 colds a year.

The U.S. Centers for Disease Control and Prevention have estimated that the average annual incidence of upper respiratory tract infections (URIs) in the United States is 429 million episodes, resulting in more than $2.5 billion in direct and indirect healthcare costs.

The common cold is most often caused by one of several hundred rhinoviruses (52%), but coronaviruses (8%) or the respiratory syncytial virus (7%) may also lead to infection. Other viruses, such as influenza (6%), parainfluenza, and adenoviruses, may produce respiratory symptoms, but these are often associated with pneumonia, fever, or chills.

Colds occur in a seasonal pattern that usually begins in mid-September and concludes in late April to early May. The common cold is quite contagious and can be transmitted by either person-to-person contact or airborne droplets. Upper respiratory symptoms usually begin 1 to 2 days after exposure and generally last 1 to 2 weeks, even though viral shedding and contagion can continue for 2 to 3 more weeks. Symptoms may persist with the occurrence of complications such as sinusitis or lower respiratory involvement such as bronchitis or pneumonia.

The common cold has a variety of overt symptoms, including malaise, nasal stuffiness, rhinorrhea, nonproductive cough, mild sore throat, and, in some cases, a low-grade fever. Because of the similarity of symptoms, a cold may be mistaken for perennial allergic rhinitis, but allergies can usually be ruled out because of the differences in chronicity.

If a patient presents with a viral URI, the spectrum of remedies is extensive. Since most of these infections are self-limiting, clinicians usually recommend rest and fluids, but other treatments include environmental and nutritional therapies, over-the-counter and prescription decongestant and antihistamine products, new antihistamine and anticholinergic nasal formulations, and antibiotics. Table 1 lists commonly used cough and cold medications and their side effects.

TABLE 1 A Profile of Common Cough and Cold Medications and their side effects Side Effects and Special Medication Purpose Considerations Aerosolized beta2 Reverse Raises heart rate and may agonists (eg, postinflammatory cause tremor albuterol) bronchospasm Alcohol-based liquid Treat multiple Potential drowsiness and combination products symptoms coordination problems Alpha1 agonists Decongestion May cause tachycardia, (oral) (eg, nervousness, transient pseudoephedrine, stimulation, dizziness, phenylpropanolamine) drowsiness, elevation of blood pressure Anticholinergic Drying May cause nasal dryness compounds: and occasional epistaxis Ipratropium bromide (topical) Other Drying May cause orthostasis, anticholinergics dysfunction of heat (eg, regulation, dry mouth, methscopolamine, constipation atropine, hyoscyamine) Antihistamines Drying Drowsiness, dry mouth, (oral) (eg, orthostatic hypertension chlorpheniramine, diphenhydramine) Benzonatate capsules Cough suppression, Chewing can numb local anesthesia the mouth; can cause sedation, dizziness Codeine, hydrocodone Cough suppression Drowsiness, constipation, nausea Dextromethorphan Cough suppression Drowsiness possible, but side effects uncommon Guaifenesin Promote No side effects; must be expectoration taken with lots of water to (mucolysis) improve efficacy Topical Decongestion Local burning; prolonged decongestants (eg, use may cause dependence oxymetazoline, phenylephrine) Zinc and vitamin C Possible reduction Possible taste disturbance, lozenges in symptom severity increase of oxalate stones and duration if susceptible

Usage of Super-compound Interferon to Prevent or Treat URI

Nearly 70˜80% URI are caused by viruses such as respiratory Syncytical virus, adenovirus, rhinovirous, cox-sackie virus, corona virus and its variant, influenza A virus and its variant, influenza B virus and its variant, parainfluenza virus and its variant, or enterovirus and its variant. A main cause of URI in adults is from rhinovirous. For children, respiratory syncytical virus and parainfluenza virus are two leading causes of URI.

Super-compound interferon plays an important role in the fight against viruses that cause URI. Super-compound interferon gains its anti-virus affects mainly via two mechanisms:

-   -   1. Attach to surface of sensitive cells and induce them to         product anti-virus protein, then block the duplication and         reproduction of viruses in vivo.     -   2. Super-compound interferon can adjust immune response,         including T-cell immune response, activity of NK cell, the         phagocytosis function of monokaryon, and even formation of some         antibodies in vivo.

In treatment for URI, Super-compound interferon can be directly applied to the affected area via a spray inspiration. This method of treatment allows the interferon to reach the target cells first hand. Consequently, marketing the supply as a spray, rather than via oral or injection, would be safer and more effective for administrating the interferon.

Usage of Super-compound Interferon to Prevent or Treat SARS

With the consent of the Sichuan working group on SARS prevention and control, the distribution of Super-compound interferon began in May of 2003. Super-compound interferon spray was allocated to doctors and nurses in hospitals, populated areas with a high risk for SARS, and to the National research group on prevention and control of SARS. Among the 3,000 users as of Dec. 19, 2003, there were no reports of any side effects connected to the use of the spray. Furthermore, none of the doctors and nurses, the people of Sichuan Province, or other organizations that have used the Super-compound interferon spray has been infected by SARS.

Therefore, this invention provides a method for inhibiting, preventing or treating virus replication or virus-infected cells by contacting said virus or infected cells with an effective amount of the super-compound interferon or its equivalent.

This super-compound interferon is useful in inhibiting, preventing or treating the following cancers or tumors:

Cancer Skin Cancer Basal Cell Carcinoma Malignant Melanoma Renal cell carcinoma Liver Cancer Thyroid Cancer Rhinopharyngeal Cancer Solid Carcinoma Prostate Cancer Stomach/Abdominal Cancer Esophageal Cancer Rectal Cancer Pancreatic Cancer Breast Cancer Ovarian Cancer & Superficial Bladder Cancer Hemangioma Epidermoid Carcinoma Cervical Cancer Non-small Cell Lung Cancer Small Cell Lung Cancer Glioma Malignant Leucocythemia Acute Leucocythemia Hemal Chronic Leucocythemia Disease Chronic Myelocytic Leukemia Hairy Cell Leukemia Lymphadenoma Multiple Myeloma Polycythemia Vera Others Kaposi's Sarcoma

Patient #1. A female patient with ovarian cancer started receiving injections. She received 15 μg injections on July 14^(th), July 16^(th), July 18^(th), July 20^(th), and July 22^(nd). On July 14^(th), 2000 ml of peritoneal fluid was observed. The patient underwent chemotherapy on July 22^(nd). On August 3^(rd), the patient's peritoneum was opened. 21 of fluid was expected to be found, but only 200 ml of fluid was observed. The left and right ovaries and lymphatic nodes were cancerous. All other organs were clear.

Patient #2. A kidney cancer patient was treated in the following manner. In a half-month period, the patient was given 3 injections of 9 μg of rSIFN-co and 3 injections of 15 μg of rSIFN-co. In the one full month following these injections, he received 9 μg and 15 μg injections of rSIFN-co every other day. A kidney biopsy showed no metastasis after this course of treatment. The patient showed a full recovery. Every half year after recovery, the patient received 15 μg injections of rSIFN-co 15 times over a one-month period.

Accordingly, this invention provides a method for inhibiting tumor or cancer cell growth by contacting the super-compound interferon or its equivalent with said tumor or cancer cells.

In a further embodiment, the super-compound interferon inhibits the DNA duplication and secretion of HBsAg and HBeAg of Hepatitis B Virus.

This invention also provides an artificial gene which codes for the super-compound interferon or its equivalent. It is within the ordinary skill to design an artificial gene. Many methods for generating nucleotide sequence and other molecular biology techniques have been described previously. See for example, Joseph Sambrook and David W. Russell, Molecular Cloning: A laboratory Manual, December 2000, published by Cold Spring Harbor Laboratory Press.

This invention provides a vector comprising the gene which codes for the super-compound interferon or its equivalent.

This invention provides an expression system comprising the vector comprising the gene which codes for the super-compound interferon or its equivalent. The cells include, but are not limited to, prokaryotic or eukaryotic cells.

This invention also provides a host cell comprising the vector comprising the gene which codes for the super-compound interferon or its equivalent.

This invention provides a process for production of recombinant super-compound interferon comprising introducing an artificial gene with selected codon preference into an appropriate host, culturing said introduced host in an appropriate condition for the expression of said compound interferon and harvesting the expressed compound interferon.

The process may comprise extraction of super-compound interferon from fermentation broth, collection of inclusion body, denaturation and renaturation of the harvested protein.

The process may maintain the high efficacy even when the super-compound interferon is used with an agent and in a particular concentration. The process also comprises separation and purification of the super-compound interferon. The process further comprises lyophilization of the purified super-compound interferon. The process comprises production of liquid injection of super-compound interferon.

This invention also provides the produced super-compound interferon by the above processes.

This invention provides a composition comprising the recombinant super-compound interferon or its equivalent and a suitable carrier.

This invention provides a pharmaceutical composition comprising the recombinant super-compound interferon or its equivalent and a pharmaceutically acceptable carrier.

This invention provides a method for treating or preventing viral diseases or tumors in a subject comprising administering to the subject an effective amount of the super-compound interferon or its equivalent.

This invention provides the above-described method wherein the viral diseases include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections of viruses caused by Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, or other type of herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia viruses I, or human T-cell leukemia viruses II, or human T-cell leukemia virus III.

This invention provides the above-described method wherein super-compound interferon was administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via an inspirator.

This invention provides the above-described method wherein super-compound interferon was administered following the protocol of injection: 9 μg or 15 μg every two days, 3 times a week, for 24 weeks.

It was surprising to find that rSIFN-co, the spatial structure of which has been changed, is not only a preparation to inhibit the DNA duplication of hepatitis B, but to inhibit the secretion of HBsAg and HBeAg on 2.2.15 cells.

One objective of this invention is to offer a preparation of rSIFN-co to directly inhibit the DNA duplication of hepatitis B viruses and the secretion of HBeAg and HBsAg of hepatitis B and decrease them to normal levels.

In one embodiment, rSIFN-co was produced with recombinant techniques. On the condition of fixed amino acid sequence, the IFN DNA was redesigned according to the E. Coli. codon usage and then the rSIFN-co gene was artificially synthesized. rSIFN-co cDNA was cloned into the high-expression vector of E. Coli. by DNA recombinant techniques, and a high expression of rSIFN-co was gained by using of induce/activate-mechanism of L-arabinose to activate the transcription of P_(BAD) promoter.

Compared with usual thermo-induction, pH induction and IPTG induction systems of genetic engineering, arabinose induction/activation system has some advantages: (1) Common systems relieve promoter function by creating a “derepression” pattern. Promoters then induce downstream gene expression. So temperature and pH change and the addition of IPTG cannot activate promoters directly. In the system disclosed herein, L-arabinose not only deactivates and represses but also activates the transcription of P_(BAD) promoter which induces a high expression of rSIFN-co. Therefore, the arabinose induction/activation system is a more effective expression system. (2) The relationship between Exogenous and L-arabinose dosage is linear. This means the concentration of arabinose can be changed to adjust the expression level of the exogenous gene. Therefore, it is easier to control the exogenous gene expression level in E. Coli. by arabinose than by changing temperature and pH value. This characteristic is significant for the formation of inclusion bodies. (3) L-arabinose is resourceful, cheap and safe, which, on the contrary, are the disadvantages of other inducers such as IPTG.

This embodiment creates an effective and resistant rSIFN-co-expressing E. Coli. engineering strain with an L-arabinose induction/activation system. The strain is cultivated and fermented under suitable conditions to harvest the bacterial bodies. Inclusion bodies are then purified after destroying bacteria and washing repeatedly. The end result, mass of high-purity, spatial-configuration-changed rSIFN-co protein for this invention and for clinical treatment, was gained from denaturation and renaturation of inclusion bodies and a series of purification steps.

The following are some rSIFN-co preparations: tablets, capsules, liquids for oral consumption, pastes, injections, sprays, suppositories, and solutions. Injections are recommended. It is common to subcutaneously inject or vein-inject the medicine. The medicine carrier could be any acceptable medicine carrier, including carbohydrates, cellulosum, adhesive, collapse, emollient, filling, add-dissolving agent, amortization, preservative, thickening agent, matching, etc.

This invention also provides a pharmaceutical composition comprising the above composition and a pharmaceutically acceptable carrier.

For the purposes of this invention, “pharmaceutically acceptable carriers” means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules, capsules, etc. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.

This invention provides a method for preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, of a subject comprising administering to the subject an effective amount of recombinant super-compound interferon or a functional equivalent thereof.

In an embodiment of the above method, the interferon is α, β, or ω.

The super-compound interferon may be administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via an inspirator.

In an embodiment, the interferon is delivered by a spray device.

In a specific embodiment, the device is described in FIG. 7.

In one of the embodiments, the interferon is lyophilized.

This invention provides a method for inhibiting the causative agent of Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, comprising contacting the agent with an effective amount of super-compound interferon or its equivalent.

It is determined that the causative agent of SARS is a virus. See eg. Rota et al (2003), Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science 1085952 and Marra, et al. (2003), The Genome Sequence of the SARS-Associated Coronavirus. Science 1085853.

This invention also provides a method for inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, or cells infected with viruses capable of inducing upper respiratory diseases, comprising contacting an effective amount of the super-compound interferon with said virus or cell. This contact could be direct or indirect.

This invention provides a composition comprising an effective amount of the super-compound interferon capable of inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, or cells infected with viruses capable of inducing upper respiratory diseases, and a suitable carrier.

This invention provides a composition comprising an effective amount of the super-compound interferon capable of preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, of a subject and a suitable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon capable of inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon capable of preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, in a subject and a pharmaceutically acceptable carrier.

This invention provides a device to deliver the above-described pharmaceutical composition.

In a preferred embodiment, the subject is a human. As it can easily be appreciated, the super-compound interferon can be used in other animals or mammals.

This invention provides a method for preventing Severe Acute Respiratory Syndrome or virus-induced upper respiratory diseases, in humans comprising application of the super-compound interferon three times a day via a spray which contains twenty micrograms of interferon, equal to ten million units of activity in three milliliter.

This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Example 1

rSIFN-co is a new interferon molecule constructed according to conservative amino acid in human IFN-α subtype with genetic engineering method. It has been proven that rSIFN-co has broad-spectrum IFN activity, such as high antivirus and tumor inhibition activity, especially for effectively treating hepatitis C.

E. Coli. codon was used to redesign rSIFN-co cDNA and then artificially synthesize cDNA of rSIFN-co from published rSIFN-co DNA sequences and deduced amino acid sequences (FIG. 1).

In order to get pure rSIFN-co protein, rSIFN-co cDNA was cloned into E. Coli. high-expression vector, and L-arabinose, which can activate strong P_(BAD) promoter in vectors, was used to induce high expression of rSlFN-co gene.

Synthesis of E. Coli. cDNA Sequence

Redesign of rSIFN-co cDNA Sequence

rSIFN-co cDNA was redesigned according to the codon usage of E. Coli. to achieve high expression in E. Coli. Deduced amino acid sequence from the redesigned cDNA sequence of rSIFN-co is completely coincidental with primitive amino acid sequence of published rSIFN-co (FIG. 1).

rSIFN-co cDNA Sequence Synthesis

rSIFN-co cDNA 5′-Terminus and 3′-Terminus Semi-Molecular Synthesis

Two semi-moleculars can be directly synthesized: rSIFN-co cDNA 5′-terminus 280 bp (fragment I) and 3′-terminus 268 bp (fragment II) by PCR. There are 41 bp overlapping among fragment II and fragment I.

(1) Chemical Synthesis Oligodeoxynucleotide Fragment:

Oligomer A: (SEQ ID NO: 5) 5′ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTG ATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGA CCGTCACGAC3′ Oligomer B: (SEQ ID NO: 7) 5′CTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGT    AACCAGTTCCAGAAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCA CAGACCTTC3′ Oligomer C: (SEQ ID NO: 8) 5′GCTGCTGGTACAGTTCGGTGTAGAATTTTTCCAGCAGGGATTCGTCCC AAGCAGCGGAGGAGTCTTTGGTGGAGAACAGGTTGAAGGTCTGCTGGATC ATTTC3′ Oligomer D: (SEQ ID NO: 9) 5′ATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGA CCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGA TGAAC3′ Oligomer B: (SEQ ID NO: 10) 5′GAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAA TACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTG CGCTTGGG3′ Oligomer F: (SEQ ID NO: 11) 5′TTATTCTTTACGACGCAGACGTTCCTGCAGGTTGGTGGACAGGGAGAA GGAACGCATGATTTCAGCACGAACAACTTCCCAAGCGCACGGGGAGTATT TTTTTTCGGTCAGG3′

PCR I for Fragment I: oligodeoxynucleotide B as template, oligodeoxynucleotide A and C as primers, synthesized 280 bp Fragment I.

PCR I mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer (Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L) 2 Oligomer A primer (25 μmol/L) 1 Oligomer C primer (25 μmol/L) 1 Oligomer B template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen American Ltd.) (25 U/μl) 1 Total volume 50 μl PCR cycle: 95 I 2 m→ (95° C.45 s→65° C.1 m→72° C.1 m) × 25 cycle→72° C.10 m→′4° C.

-   -   PCR II for Fragment II: oligodeoxynucleotide E as template,         oligodeoxynucleotide D and F as primers, synthesized 268 bp         Fragment II.

PCR II mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer (Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L) 2 Oligomer D primer (25 μmol/L) 1 Oligomer F primer (25 μmol/L) 1 Oligomer E template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen American Ltd.) 1 (25 U/μl) Total volume 50 μl PCR cycle: the same as PCR I

Assembling of rSIFN-co cDNA

Fragment I and II were assembled together to get the complete cDNA molecular sequence of rSIFN-co using the overlapping and extending PCR method. Restriction enzyme Nde I and Pst I were introduced to clone rSIFN-co cDNA sequence into plasmid.

-   -   (1) Chemical synthesis primers

Oligomer G: (SEQ ID NO: 12) 5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′ Oligomer H: (SEQ ID NO: 13) 5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′

-   -   (2) Overlapping and extending PCR

PCR mixture (units: μl) sterilized distilled water 38 10 × Pfu buffer (Stratagen American Ltd.)  5 dNTP mixture (dNTP concentration 2.5 mmol/L)  2 primer G (25 μmol/L)  1 primer H (25 μmol/L)  1 *fragment I preduction (1 μmol/L)  1 *fragment II preduction (1 μmol/L)  1 Pfu DNA polymerase (Stratagen American Ltd.) (2.5 U/μl)  1 Total volume 50μ *Separate and purify PCR production with StrataPrep PCR purification kit produced by Stratagen American Ltd. And dissolve into sterilized distilled water. PCR cycle: the same as PCR I

rSIFN-co Gene Clone and Sequence Analysis

pLac T7 plasmid as cloning vector. pLac T7 plasmid is reconstructed with pBluescript II KS(+) plasmid produced by Stratagen (FIG. 3).

Purified PCR production of rSIFN-co cDNA with StrataPrep PCR purification kit. Digest cDNA and pLac T7 plasmid with NdeI and PstI. Run 1% agarose gel electrophoresis and separate these double-digested DNA fragments. Recover 507 bp long rSIFN-co DNA fragment and 2.9 kb plasmid DNA fragment. Ligate these fragments by T4 DNA ligase to form a recombinant plasmid. Transform DH_(5α)competent cells (Gibco) with the recombinant plasmid, culture at 37° C. overnight. Identify the positive recombinant colony, named pHY-1.

Run DNA sequencing with SequiTherm™ Cycle Sequencing Kit produced by American Epicentre Technologies Ltd using L1-COR Model 4000L. Primers are T7 and T3 common sequence primer, the DNA sequencing result matches theoretic design.

Purify the rSIFN-co, sequence the N-terminus amino acids, the N-terminus amino acid sequence matches experimental design which is as follows:

(SEQ ID NO: 14) N-Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn-Arg- Arg-Ala-Leu

Construction, Transformation, Identification, and Hereditary Stability of Expression Vector

Construction and Transformation of Expression Vector

Digested E. Coli. expression vector pHY-4 (see FIG. 3) with Nde I to linearize and subsequently digest with Xba I. Run 1% agarose gel electrophoresis, and purify the 4.8 kb pHY-4 Nde I-Xba I digest fragment with QIAEX II kit produced by QIAGEN Germany Ltd.

At the same time, the pHY-4 plasmid is double digested with Nde I-Xba I. Run 1% agarose gel electrophoresis and purify the 715 bp fragment. Ligate the rSIFN-co and pHY-4 fragments with T4 DNA ligase to construct the recombinant plasmid (See FIG. 4). Transform DH_(5α)competent cells with the recombinant plasmid. Spread the transformed cells on LB plate with Amp, 37° C. culture overnight.

Positive Cloning Strain Screening

Randomly choose E. Coli. colonies from above LB-plate, screening the positive strains containing recombinant vector by endonuclease digesting and PCR analysis. Name one of the positive recombinant plasmid pHY-5, and name the strain containing pHY-5 plasmid PVIII. Amplify and store the positive strain with glycerol in −80° C.

High Expression of rSIFN-co Gene in E. Coli.

In pHY-5 plasmid, rSIFN-co gene is under control of strong promoter P_(BAD). This promoter is positively and negatively regulated by the product of the gene araC. AraC is a transcriptional regulator that forms a complex with arabinose. In the absence of arabinose, the AraC dimer binds O₂ and I₁ forming a 210 bp loop. This conformation leads to a complete inhibition of transcription. In the presence of arabinose, the dimer is released from O₂ and binds I₁ and I₂ leading to transcription. Arabinose binding deactivates, represses and even activates the transcription of P_(BAD) promoter, which stimulates P_(BAD) inducing high expression of rSIFN-co. rSIFN-co expression level in PVIII is more than 50% of the total E. Coli. protein.

SUMMARY

RSIFN-CO is a new interferon molecule artificially built according to the conservative amino acid of human α interferons. It has been proven as a effective anti-hepatitis drug. In order to get enough pure rSIFN-co protein, a stable recombinant E. Coli. strain which high expresses rSIFN-co protein was constructed.

First, according to published rSIFN-co amino acid sequence, E. Coli. codon was used to synthesize whole cDNA of rSIFN-co. This DNA fragment was sequenced and proved that the 501 bp codon sequence and TAA termination codon sequence are as expected and consistent with the experimental design. Subsequent analysis revealed that the N-terminus amino acid sequence and amino acid composed of rSIFN-co produced by the recombinant strain were both consistent with the prediction.

The rSIFN-co cDNA was cloned into E. Coli. high-expression vector pHY-4 plasmid to construct the recombinant plasmid pHY-5. E. Coli. LMG194 strain was further transformed with pHY-4 plasmid to get stable rSIFN-co high-expression transformant. This transformant was cultured for 30 generations. The heredity of pHY-5 recombinant plasmid in E. Coli. LMG194 was normal and stable, and the expression of rSIFN-co was high and steady.

E. Coli. LMG194, which contains recombinant pHY-5 plasmid, is actually an ideal high-expression engineering strain.

REFERENCES

1. Blatt L M, Davis J M, Klein S B. et al. The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon. Journal of Interferon and Cytokine Research, 1996;16(7):489-499.

2. Alton, K. et al: Production characterization and biological effects of recombinant DNA derived human IFN-α and IFN-γ analogs. In: De Maeger E, Schellekens H. eds. The Biology of Interferon System. 2nd ed. Amsterdam: Elsevier Science Publishers, 1983: 119-128

3. Pfeffer L M. Biologic activity of natural and synthetic type 1 interferons. Seminars in Oncology, 1997;24 (3 suppl 9):s9-63-S9-69.

4. Ozes O N, Reiter Z, Klein S, et al. A comparison of interferon-con1 with natural recombinant interferons-(: antiviral, antiproliferative, and natural killer-inducing activities. J. Interferon Res., 1992; 12:55-59.

5. Heathcote E J L, Keeffe E B, Lee S S, et al. Re-treatment of chronic hepatitis C with consensus interferon. Hepatology, 1998;27(4):1136-1143.

6. Klein M L, Bartley T D, Lai P H, et al. Structural characterization of recombinant consensus interferon-alpha. Journal of Chromatography, 1988; 454:205-215.

7. The Wisconsin Package, by Genetics Computer Group, Inc. Copyright 1992, Medison, Wis., USA

8. Nishimura, A et al: A rapid and highly efficient method for preparation of competent E. coli cells. Nuclei. Acids Res. 1990, 18:6169

9. All molecular cloning techniques used are from: Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A laboratory manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbour, N.Y. 1989.

10. Guzman, L. M et al: Tight regulation, modulation, and high-level express-ion by vectors containing the arabinose PBAD promoter. J. Bacteriol. 1995, 177: 4121˜4130.

rSIFN-co cDNA Sequence Designed According to E. COLI. Codon Usage and Deduced rSIFN-co Amino Acid Sequence

5′   11    21    31   41   51 +1 M C D L P Q T H S L G N R R A L I L L A 1 ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCT TACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA 5′   71    81    91   101   111 +1 Q M R R I S P F S C L K D R H D F G F P 61 CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCG GTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC 5′   131   141   151   161   171 +1 Q E E F D G N Q F Q K A Q A I S V L H E 121 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAA GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCA AGACGTGCTT 5′   191   201   211   221   231 +1 M I Q Q T F N L F S T K D S S A A W D E 181 ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAA TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT 5′   251   261   271   281   291 +1 S L L E K F Y T F L Y Q Q L N D L F A C 241 TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGC AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGA CCTTCGAACG 5′      311   321      331   341   351 +1 V I Q E V G V E E T P L M N V D S I L A 301 GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCT CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA 5′     371   381    391   401   411 +1 V K K Y F Q R I T L Y L T E K K Y S P C 361 GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGC CAATTTTTTA TGAAGGTCGC ATAGTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG 5′     431   441    451   461   471 +1 A W E V V R A E I M R S F S L S T N L Q 421 GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG CGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC 5′    491    501 (SEQ ID NO: 12) +1 E R L R R K E # 481 GAACGTCTGC GTCGTAAAGA ATAA CTTGCAGACG CAGCATTTCT TATT

Example 2

Separation and Purification of rSIFN-co

1. Fermentation

Inoculate the recombinant strain in LB media, shaking (200 rpm) under 37° C. overnight (approximate 18 h), then add 30% glycerol to the fermentation broth to get final concentration of 15%, allotted to 1 ml tube and kept in −20° C. as seed for production.

Add 1% of the seed to LB media, shaking (200 rpm) under 37° C. overnight to enlarge the scale of the seed, then add to RM media with a ratio of 10%, culturing under 37° C. Add arabinose (20% solution) to 0.02% as an inductor when the OD600 reaches about 2.0. 4 hours after that, stop the culture process, collect the bacteria by centrifuge, resuspend the pellet with buffer A, and keep in −20° C. overnight. Thaw and break the bacteria by homogenizer, then centrifuge. Wash the pellet with buffer B, buffer C, and distilled water to get a relatively pure inclusion body.

2. Denaturation and Renaturation

Dissolve the inclusion body in Guanidine-HCl (or urea) of 6 mol/L. The solution will be a little cloudy. Centrifuge it at a speed of 10000 rpm. Determine the protein concentration of the supernatant. This supernatant is called “denaturation solution.” Add the denaturation solution to renaturation buffer, and keep the final protein concentration under 0.3 mg/ml. It is better to add the totally denaturation solution in three steps instead of one step. Keep the solution overnight under 4° C. Afterwards, dialyze 10 mol/L, 5 mol/L PB buffer and distilled water, then adjust its pH by 2 mol/L HAc-NaAc. Let it stand, then filtrate.

3. Purification

-   -   POROS HS/M anion exchange chromatography:

Chelating sepharose™ fast flow: Add PB buffer of 0.2 mol/L (pH 6.6) and NaCl of 4 mol/L in the solution from HS to adjust solution pH to pH 6.0 and NaCl concentration to 1 mol/L.

Condense the eluted solution by POROS HS/M. Sometimes a purification by sephacryl S-100 step can be added to meet stricter purity requirements.

Note:

-   -   Buffer A: 100 mmol/L Tris-HCl, pH 7.5-10 mmol/L EDTA-100 mmol/L         NaCl     -   Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L         EDTA-0.5% Triton X-100     -   Buffer C: 50 mmol/L Tris-HCl, pH 7.5-2 mol/L Urea-10 mmol/L         EDTA-0.5% Triton X-100     -   Buffer D: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 5.5)     -   Buffer E: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 5.0)     -   Buffer F: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 4.0)     -   Buffer G: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 3.6)     -   Renaturation buffer: 0.5 mol/L Arginine-150 mmol/L Tris-HCl, pH         7.5-0.2 mmol/L EDTA

LB Media: 1 L

Tryptone 10 g Yeast extracts  5 g NaCl 10 g

RM Media: 1 L

Casein 20 g MgCl 1 mmol/L (0.203 g) Na₂HPO₄ 4 g; KH₂PO₄ 3 g, NaCl 0.5 g NH₄Cl 1 g

After purification, the buffer was changed to PBS (pH 7.0) along with the step of condensing by POROS HS/M. This is called the “Protein Stock Solution.” It can directly used in the preparation of injections or sprays, or stored at 2-8° C.

Formula for Injection:

Solution Lyophilized powder Solution of rSIFN-co 34.5 μg/ml 34.5 μg/ml PB (pH 7.0) 25 mmol/L 10 mmol/L Glycine — 0.4 mol/L NaCl 0.1 mol/L —

For Spray:

EDTA 0.01% Tween 80 0.05% Trisodium citrate 10 mmol/L Glycerol 1.26% Sodium Chloride 0.03% Phenylmethanol  0.5% HSA  0.1% rSIFN-co 10 μg/ml

Quality Control Process

During purification, tests for protein content, protein purity, specific activity, and pyrogen are conducted after each step. When the stock solution is obtained, all the tests listed in the table are done one after the other.

The quality of the product is controlled according to “Chinese Requirements for Biologics”

1. Original Protein Solution

Lowry Item of Test Method Protein Stock Solution: Test for Protein Content Lowry Test for Protein Purity Non-reductive SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) HPLC Analysis Test for Molecular Weights Reductive SDS-PAGE Test for Specific Activity According to Method in “Specific Activity Test of Interferon Test for Leftover Exogenetic Using DNA Labeling and DNA Detection Kit Test for Activity of According to Method in Leftover Antibiotics “Chemical and Other Test Methods for Biologics” Test for Bacterial Endotoxin According to Method in “Requirements for Bacterial Endotoxin Test of Biologics” Test for Isoelectronic Point Isoelectric Focusing Electrophoresis Test for Identify UV spectrum (range of Characteristics of the wavelength: 190-380 nm) Protein Peptide Mapping (hydrolyzed by pancreatic enzyme, analyzed by C-18 column) N-terminal Sequence Test C-terminal Sequence Test Circular Dichroism Amino Acid Analysis Semi-finished Product Test for Bacterial Endotoxin According to Method in “Requirements for Bacterial Endotoxin Test of Biologics” Product Appearance Check Chemical According to Method in “Chemical and Other Test Methods for Biologics” Test for Specific Activity According to Method in “Specific Activity Test of Interferon Sterility Test According to Method in “c” Abnormal Toxicity Test Test on Mouse Pyrogen Test According to Method in “Requirements for Pyrogen Test of Biologics” Test for Stability of Product Note: “Chemical and Other Test Methods for Biologics”, “Requirements for Pyrogen Test of Biologics” and “Requirements for Bacterial Endotoxin Test of Biologics” all can be found in the “Chinese Requirements for Biologics.” “Chinese Requirements for Biologics, ” PAN Zhengan, ZHANG Xinhui, DUAN Zhibing, et al. Chinese Biologics Standardization committee. Published by Chemical Industry Publishing Company, 2000.

Example 3 Stability of Lyophilized Powder of Recombinant Super-Compound Interferon Injection

The stability experiments were carried out with samples of lyophilized powder of recombinant super-compound interferon (rSIFN-co) injection in two specifications and three batches. The experiments started on April, 2000.

1. Sample Source

Samples were supplied by Sichuan Huiyang Life-engineering Ltd., Sichuan Province. Lot: 990101-03, 990101-05, 990102-03, 990102-05, 990103-03, 990103-05

2. Sample Specifications

Every sample in this experiment should conform with the requirements in the table below.

TABLE 3.1 Standard of Samples in Experiment Items Standards 1. Appearance white loose powder 2. Dissolving dissolve rapidly in injection water time (within 2 min) at room temperature 3. Clarity colorless liquid or with little milk- like glisten; should not be cloudy, impurity or with indiscernible deposit 4. pH value 6.5~7.5 5. Potency (IU/dose) 80%~150% of indicated quantity (9 μg: 4.5 × 10⁶ IU, 15 μg: 7.5 × 10⁶ IU) 6. Moisture no more than 3.0% (W/W)

3. Experimental Content

Test samples at 2˜8° C.: The test samples were put into a 2˜8° C. refrigerator, then the above items of these samples were respectively tested in the 1^(st,), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th), 30^(th), 36^(th) month. The results were recorded.

Test samples at 25° C.: The test samples were put into a thermostat at 25° C., then the above items of these samples were respectively tested in the 1^(st,), 3^(rd), 6^(th), 9^(th), 12th, 18^(th), 24^(th), 30^(th) month. The results were recorded.

Test samples at 37° C.: The test samples were put into a thermostat at 37° C., then the above items of these samples were respectively tested in the 1^(st,), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th) month. The results were recorded.

4. Results and Conclusion

-   -   1) At 37° C., according to data collected at designated points         during testing and compared with data before testing, the         potency began descending from the 6^(th) month and the changes         in the three batches were similar. The appearance of other items         had no changes.     -   2) At 25° C., according to data collected at designated points         during testing and compared with data before the testing, the         potency only had a little change, and the changes in the three         batches were similar. The appearance of other items had no         changes.     -   3). At 2-8° C., according to data collected at designated points         during testing and compared with data before testing, the         potency of the three batches all were stable. The appearance of         other items also had no changes.     -   In conclusion, it is suggested that the lyophilized powder of         recombinant super-compound interferon for injection should be         better stored and transported at low temperatures. Without such         conditions, the product can also be stored for short periods         (i.e. 3 months) at room temperature.

Example 3.5 Production Flow Chart of rSIFN-co

1. Production

1.1 Fermentation

-   -   Use mixture of LB+M9 as culturing medium. The amount of         innoculum will be 1.5%. Agitate to OD600=0.4 (about 3.5 hours)         under 32° C., then raise temperature to 42° C. Continue the         agitation for another 6 hours, the expression of rSIFN-co will         reach the maximum level. The examination under scanning of the         gel resulting from SDS-PAGE shows that the level of expression         is up to 57%, which is the highest standard in China.

1.2 Purification

The purity of the product (rSIFN-co) from this production procedure is shown to 95% under the test of SDS-PAGE where molecular weight is 14.5 Kda. The reverse phase HPLC shows a single peak and the purity is up to 97%. Its specific activity is up to 1×10⁹ IU/mg protein.

1.3 Packaging and Inspection

-   -   After HPLC purification, 2% human serum albumin, 1% sucrose and         1% glucose are added to the rSIFN-co. It is then separated and         lyophilized into injection sample. When tested under the         Wish-VVS inspection system, the result was 4.5×10⁸ IU. When         tested with aseptic inspection and pyrogen inspection under the         standard requirement of China, the results were negative. This         result complies with the requirements for IV injection.

2. Quality Control

2.1 Biological Characteristics

-   -   (1) When using LB+M9 to cultivate bacteria, the characteristics         should match with the typical characteristics of E-coli         bacteria. No other bacteria were detected.     -   (2) When smeared for Gram staining and inspected under a         microscope, it is bacteria-negative.     -   (3) Reaction to antibiotics is the same as those original         bacteria.     -   (4) Electron microscope inspection shows typical characteristics         of E-coli bacteria. No mycoplasma, virus spore or other micro         pollutes was detected.     -   (5) Biochemical reaction test shows characteristics of E-coli         bacteria.

2.2 Quality Control of Interferon Expression

-   -   (1) Interferon expression (cultivated in an agitating platform)         matches the amount of expression in original input bacteria.     -   (2) When tested with anti-interferon serum, a reaction is shown.     -   (3) Plasmid inspection: Restriction digest matched with the         original plasmid.

2.3 Bacteria Strain Product

-   -   Bacteria strain product denotes the specimen from the original         bacteria strain that was produced from the procedures shown in         1.2.     -   The bacteria strain product should be inspected as follows to         make sure there is no derivation: Use LB to plate 2-3 pieces and         cultivate. Separate and take 5-10 bacteria groups for the test         of interferon expression. Repeat the test at least two (2)         times. Only use the one which shows the highest % to be the         bacteria strain product.

2.4 Innoculum

-   -   The innoculum denotes the chosen bacteria strain product after         fermentation. The amount, cultivation time and most appropriate         OD value of innoculum can be decided according to bacteria         strain. An anti-polluted bacteria procedure should apply for         whatever innoculum would be produced.

2.5 Growing of Bacteria Strain

-   -   Growing of bacteria strain would be done in a Bacteria Free room         environment where no more than one bacterium is growing in the         same room. Same culturing medium will be used for both bacteria         strain and innoculum. The one used in rSIFN-co is LB.

2.6 Fermentation

-   -   (1) Fermentation only takes place in a clean fermentation room         with a single bacteria fermentation environment.     -   (2) Cleaning of fermentation container and tube is done twice,         before and after the insertion of culturing medium. Then, the         container should be frozen to reach the appropriate temperature         for innoculum.     -   (3) Avoid using antibiotic which might affect cell growth in the         culturing medium.     -   (4) Fermentation parameters like temperature, pH value,         dissolved oxygen and time required could be varied according to         different types of bacterial strains.

2.7 Bacteria Collection

-   -   (1) Centrifuge the bacteria solution to collect bacteria or use         another method. All apparatus should be cleaned before and after         the operation. The waste solution should be drained after the         cleaning procedure.     -   (2) The bacteria should be kept under 4-8° C. if they are going         to be split within 24 hours. Otherwise, they should be kept         under −30° C. Those are kept under such conditions can be used         within 6 months.

2.8 Bacteria Cell Lysis

-   -   (1) Use appropriate buffer solution to balance the bacteria         strain. Cell lysis can be done by physical, chemical or         biological methods. Use centrifuge to precipitate the bacteria         and apply cleaning solutions.     -   (2) If the chemical method is used to split cells, no solutions         harmful to human beings should be used.

2.9 Purification

-   -   (1) Purification will get rid of most of the non-interferon         contents. In the process of purification, no toxic materials         should be found if extra elements are added.     -   (2) If using antibody affinity chromatography for purification,         there should be an indication of the source and degree of         purity. Also, inspection of small quality IgG should be         performed.     -   (3) During the process of purification, clearance of pyrogen is         critical. All apparatus should be checked to eliminate this         interference.     -   (4) The highly concentrated interferon is known as “intermediate         product”. After inspection and tests, add albumin to raise the         concentration to 2% which is now known as “albumin intermediate         product”. After examination and tests, it should be kept at         −30° C. and never thawed before use. This product should be used         within 6 months.     -   (5) The albumin that is used in this process should also fulfill         tests and requirements such as: negativity under RBSAG         inspection and an indication of the ratio among monomer, dimer         and polymer.

2.10 Production into Tube Product

-   -   (1) Filtration: Use 0.22μ membrane to filter the bacteria. The         product should be handled with aseptic techniques. Samples         should be taken to test the value of the interferon.     -   (2) Dilution: Dilute the albumin intermediate product with 2%         diluent. No preservative should be added. The product can be         lyophilized after the aseptic inspection and pyrogen inspection.

2.11 Lyophilization

-   -   The lyophilization should not affect the activity of interferon,         and the water content of said lyophilite will be maintained.

2.12 Inspection

-   -   There are two types of rSIFN-co made. One is for injection and         the other for topical use. The specifications for the two are         different. There are intermediate products and final products         for each type. In the injection type, intermediate products         include purified interferon, albumin intermediate product, and         bacteria free albumin intermediate product. Final product from         the injection type will denote only lyophilized product. The         intermediate product in the topical type denotes only purified         interferon. The final product from the topical type denotes only         separated packed liquid formed lyophilized products.

2.13 Packaging

-   -   There is different packaging for the injection type and the         topical type.

2.14 Storage

-   -   The product should be kept at 4° C. The purification solution         should not be stored in a frozen state.

2.15 Expiration

-   -   The expiration period is two (2) years after the lyophilization         procedure for lyophilized products. The expiration period is 6         months after individual packing for liquidated products.

Example 4 rSIFN-co Inhibits HBV-DNA Duplication and Secretion of HBsAg and HBeAg

Materials

Solvent and Dispensing Method: Add 1 ml saline into each vial, dissolve, and mix with MEM culture medium at different concentrations. Mix on the spot.

Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased from Schering Plough. 3×10⁶ U each, mix to 3×10⁶ IU/ml with culture medium; Infergen® (liquid solution), purchased from Amgen, 9 μg, 0.3 ml each, equal to 9×10⁶ IU, and mix with 9×10⁶ IU/ml culture medium preserve at 4° C.; 2.2.15 cell: 2.2.15 cell line of hepatoma (Hep G2) cloned and transfected by HBV DNA, constructed by Mount Sinai Medical Center.

Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum, HycloneLab American Ltd. G-418 (Geneticin); MEM dispensing, Gibco American Ltd.; L-Glutamyl, imported and packaged by JING KE Chemical Ltd.; HBsAg and HBeAg solid-phase radioimmunoassay box, Northward Reagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern China Medicine; And Lipofectin, Gibco American Ltd.

Experimental goods and equipment: culture bottle, Denmark Tunclon™; 24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxide hatching box, Shel-Lab American Ltd.; MEM culture medium 100 ml: 10% cattle fetal blood serum, 3% Glutamy 11%, G418 380 μg/ml, biograncetina 50 U/ml.

Method:

2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box with full of 2.2.15 cell, digest at 37° C. for 3 minutes, and add culture medium to stop digest and disturb it to disperse the cells, reproduce with ratio of 1:3. They will reach full growth in 10 days.

Toxicity test: Set groups of different concentrations and a control group in which cell is not acted on with medicine. Digest cell, and dispense to a 100,000 cell/ml solution. Inoculate to 96-well culture board, 200 μl each well, culture at 37° C. for 24 h with 5% CO₂. Test when simple cell layer grows.

Dispense rSIFN-co to 1.8×10⁷ IU/ml solution than prepare a series of solutions diluted at two-fold gradients. Add into 96-well culture board, 3 wells per concentration. Change the solution every 4 days. Test cytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as 3, 50% as 2, 25% as 1, zero as 0. Calculate average cell lesion and inhibition rate of different concentrations. Calculate TC50 and TC0 according to the Reed Muench method.

${{TC}\; 50} = {{Antilog}\left( {B + {\frac{50 + B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration, C=log dilution power

Inhibition test for HBeAg and HBsAg: Separate into positive and negative HBeAg and HBsAg contrast groups, cell contrast group and medicine concentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into 6-well culture board, 3 ml each well, culture at 37° C. for 24 h with 5% CO₂, then prepare 5 gradiently diluted solutions with 3-fold as the grade (Prepare 5 solutions, each with a different protein concentration. The concentration of Solution 2 is 3 times lower than that of Solution 1, the concentration of Solution 3 is 3 times lower than that of Solution 2, etc.) 4.5×10⁶ IU/ml, 1.5×10⁶ IU/ml, 0.5×10⁶ IU/ml, 0.17×10⁶ ¹ U/ml, and 0.056×10⁶ ¹ U/ml, 1 well per concentration, culture at 37° C. for 24 h with 5% CO₂. Change solutions every 4 days using the same solution. Collect all culture medium on the 8^(th) day. Preserve at −20° C. Repeat test 3 times to estimate HBsAg and HBeAg with solid-phase radioimmunoassay box (Northward Reagent Institute of Chinese Isotope Ltd.). Estimate cpm value of each well with a γ-accounting machine.

Effects calculation: Calculate cpm mean value of contrast groups and different-concentration groups and their standard deviation, P/N value such as inhibition rate, IC50 and SI.

-   -   1) Antigen inhibition rate

$(\%) = {\frac{A - B}{A} \times 100}$

-   -   -   A=cpm of control group; B=cpm of test group;

    -   2) Counting the half-efficiency concentration of the medicine         -   Antigen inhibition

${{IC}\; 50} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration, C=log dilution power

-   -   3) SI of interspace-conformation changed rSIFN-co effect on         HBsAg and HBeAg in 2.2.15 cell culture:

${S\; I} = \frac{{TC}\; 50}{{IC}\; 50}$

-   -   4) Estimate the differences in cpm of each dilution degree from         the control group using student t test

Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days. Exsuction culture medium (Separate cells from culture medium by means of draining the culture medium.). Add lysis buffer to break cells, then extract 2 times with a mixture of phenol, chloroform and isoamyl alcohol (1:1:1), 10,000 g centrifuge. Collect the supernatant adding anhydrous alcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20 μlTE buffer. (2) Electrophoresis: Add 6×DNA loading buffer, electrophoresis on 1.5% agarose gel, IV/cm, at fixed pressure for 14-18 h. (3) Denaturation and hybridization: respectively dip gel into HCl, denaturaion buffer and neutralization buffer. (4) Transmembrane: Make an orderly transfer of DNA to Hybond-N membrane. Bake, hybridize and expose with dot blot hybridization. Scan and analyze relative density with gel-pro software. Calculate inhibition rate and IC50.

Results

Results from Tables 4.1, 4.2 and 4.3 show: After maximum innocuous concentration exponent culturing for 8 days with 2.2.15 cell, the maxima is 9.0±0×10⁶ IU/ml average inhibition rate of maximum innocuous concentration rSIFN-co to HBeAg is 46.0±5.25% (P<O. 001), IC50 is 4.54±1.32×10⁶ IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC50 is 6.49±0.42×10⁶ IU/ml, SI is 2.77. This shows that rSIFN-co can significantly inhibit the activity of HBeAg and HBsAg, but that the IFN of the contrast group and Infergen® cannot. It has also been proved in clinic that rSIFN-co can decrease HBeAg and HBsAg or return them to normal levels.

TABLE 4.1 Results of inhibition rate of rSIFN-co to HBsAg and HBeAg Inhibition rate Average Concentration Second First Second Third inhibition Accumulated (×10⁴ IU/ml) First well well Third well well well well rate Accumulation 1-Accumulation inhibition rate First batch: (rSIFN-co) Inhibition effect to HBeAg 900 9026 8976 10476 0.436227 0.43935 0.345659 0.407079 0.945909 0.592921 0.614693546 300 9616 12082 10098 0.3993754 0.245347 0.369269 0.337997 0.5388299 1.254924 0.300392321 100 9822 16002 12800 0.386508 0.0005 0.2005 0.195836 0.200833 2.059088 0.08867188  33.33333 15770 19306 16824 0.014991 0 0 0.004997 0.0049969 3.054091 0.001633453  11.11111 19172 22270 18934 0 0 0 0 0 4.054091 0 Control Cell 16010 Blank 0 Dilution 3 IC50 602.74446016 Inhibition effect to HBsAg 900 7706 7240 7114 0.342155 0.381936 0.392693 0.372261 0.922258 0.627739 0.595006426 300 8856 7778 9476 0.2439816 0.336008 0.191053 0.257014 0.5499972 1.370724 0.286349225 100 10818 10720 10330 0.07649 0.084856 0.118149 0.093165 0.292983 2.27756 0.113977019  33.33333 10744 11114 10570 0.082807 0.051221 0.097661 0.07723 0.1998179 3.20033 0.058767408  11.11111 10672 9352 10810 0.088953 0.201639 0.077173 0.122588 0.122588 4.077742 0.02918541 Control Cell 11714 Blank 0 Dilution 3 IC50 641.7736749 Second batch: (rSIFN-co) Inhibition effect to HBeAg 900 7818 8516 9350 0.554378 0.514592 0.467054 0.512008 1.371181 0.487992 0.737521972 300 10344 10628 9160 0.4103967 0.394209 0.477884 0.427497 0.8591731 1.060496 0.447563245 100 12296 14228 13262 0.299134 0.18901 0.244072 0.244072 0.4316522 1.816423 0.19201839  33.33333 15364 17414 16188 0.124259 0.00741 0.77291 0.069653 0.1876045 2.74677 0.063933386  11.11111 17386 13632 15406 0.009006 0.222982 0.121865 0.117951 0.117951 3.628819 0.03148073 Control Cell 16962 Blank 0 Dilution 3 IC50 365.9357846 Inhibition effect to HBsAg 900 5784 6198 5792 0.498265 0.462353 0.497571 0.486063 0.893477 0.513937 0.634835847 300 7150 8534 8318 0.379771 0.259715 0.278452 0.30598 0.4074138 1.207957 0.252210647 100 9830 11212 10210 0.147294 0.027412 0.11433 0.096345 0.101434 2.111612 0.04583464  33.33333 13942 12368 13478 0 0 0 0 0.0050891 3.111612 0.001632835  11.11111 12418 11634 11352 0 0 0.015267 0.005089 0.005089 4.106523 0.001237728 Control Cell Blank 0 Dilution 3 IC50 611.0919568 Third batch: (rSIFN-co) Inhibition effect to HBeAg 900 9702 9614 8110 0.428016 0.433204 0.521872 0.461031 1.316983 0.538969 0.709599543 300 8914 10032 8870 0.4744723 0.40856 0.477066 0.453366 0.8559525 1.085603 0.440859127 100 16312 12688 13934 0.038321 0.251975 0.178517 0.156271 0.402586 1.929332 0.172641621  33.33333 15080 12814 13288 0.110954 0.244547 0.216602 0.190701 0.2463153 2.738631 0.082519158  11.11111 21928 15366 15728 0 0.094093 0.072751 0.0055615 0.055615 3.683017 0.014875633 Control Cell 17544 Blank 0 Dilution 3 IC50 382.0496935 Inhibition effect to HBsAg 900 5616 6228 5346 0.496864 0.442035 0.521054 0.486651 0.763125 0.513349 0.597838293 300 8542 8590 7096 0.234725 0.230425 0.364272 0.276474 0.2764738 1.236875 0.182690031 100 11420 11360 11394 0 0 0 0 0 2.236875 0  33.33333 12656 11582 13110 0 0 0 0 0 0  11.11111 13142 12336 13342 0 0 0 0 0 4.236875 0 Control Cell 11528 Blank 0 Dilution 3 IC50 694.7027149 HBeAg: Average IC50: 450.2434 SD: 132.315479 HBsAg: Average IC50: 649.1894 SD: 42.29580

TABLE 4.2 Results of inhibition rate of Intron A(IFN-α2b) to HBsAg and HBeAg Con- Inhibition rate Average Accumulated centration First Second Third First Second Third inhibition 1- inhibition (×10⁴ IU/ml) well well well well well well rate Accumulation Accumulation rate Inhibition effect to HBeAg 300 14918 11724 9950 0 0.029711 0.176529 0.068747 0.068747 0.931253 0.068746724 100 14868 16890 15182 0 0 0 0 0 1.931253 0  33.33333 16760 21716 16400 0 0 0 0 0 2.931253 0  11.11111 20854 15042 16168 0 0 0 0 0 3.931253 0  3.703704 12083 12083 12083 0 0 0 0 0 4.931253 0 Control Cell 17544 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 300 9226 8196 9658 0.152489 0.247106 0.521054 0.1708 0.189295 0.8292 0.185857736 100 10946 10340 10828 0 0.050156 0.364272 0.018495 0.0184947 1.810705 0.010110817  33.33333 12250 12980 13934 0 0 0 0 0 2.810705 0  11.11111 12634 12342 12000 0 0 0 0 0 3.810705 0  3.703704 10886 10886 10886 0 0 0 0 0 4.810705 0 Control Cell 10886 Blank 0 Dilution 3 IC50 FALSE

TABLE 4.3 Results of inhibition rate of Infergen ® to HBsAg and HBeAg Con- Inhibition rate Average Accumulated centration First Second Third First Second Third inhibition 1- inhibition (×10⁴ IU/ml) well well well well well well rate Accumulation Accumulation rate First batch: (Infergen ®) Inhibition effect to HBeAg 900 14172 12156 17306 0.091655 0.220869 0 0.104175 0.306157 0.895825 0.254710274 300 13390 12288 16252 0.1417767 0.212409 0 0.118062 0.2019827 1.777764 0.102024519 100 14364 18834 14194 0.079349 0 0.090245 0.056531 0.083921 2.721232 0.029916678 33.33333 15722 16034 16340 0 0 0 0 0.0273897 3.721232 0.007306592 11.11111 17504 17652 14320 0 0 0.082169 0.02739 0.02739 4.693843 0.005801377 Control Cell 15602 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 12080 11692 12234 0 0.01275 0 0.00425 0.025163 0.99575 0.024647111 300 12840 11484 12350 0 0.030313 0 0.010104 0.0209125 1.985646 0.010422073 100 12894 14696 15086 0 0 0 0 0.010808 2.985646 0.003606955  33.33333 15032 12928 13020 0 0 0 0 0.0108081 3.985646 0.002704416  11.11111 11794 11984 11508 0.004137 0 0.028287 0.010808 0.010808 4.974837 0.002167838 Control Cell 11843 Blank 0 Dilution 3 IC50 FALSE Second batch: (Infergen ®) Inhibition effect to HBeAg 900 6278 6376 6408 0.200051 0.187564 0.183486 0.190367 0.274635 0.809633 0.253290505 300 7692 9092 6394 0.0198777 0 0.18527 0.068383 0.0842678 1.74125 0.046161005 100 8960 7474 8190 0 0.047655 0 0.015885 0.015885 2.725365 0.005794856  33.33333 8530 8144 9682 0 0 0 0 0 3.725365 0  11.11111 7848 7848 7848 0 0 0 0 0 4.725365 0 Control Cell 7848 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 12364 12268 12274 0.036171 0.043655 0.043187 0.041004 0.140162 0.958996 0.12751773 300 11590 12708 13716 0.0965076 0.009355 0 0.035287 0.0991581 1.923709 0.0490186 100 12448 13468 13982 0.029623 0 0 0.009874 0.063871 2.913834 0.02144964  33.33333 12616 11346 12444 0.016526 0.115529 0.029935 0.053996 0.0539965 3.859838 0.013796309  11.11111 12828 12828 12828 0 0 0 0 0 4.859838 0 Control Cell 12828 Blank 0 Dilution 3 IC50 FALSE Third batch: (Infergen ®) Inhibition effect to HBeAg 900 7240 6642 6158 0.064599 0.14186 0.204393 0.136951 0.217399 0.863049 0.201211735 300 11072 8786 6902 0 0 0.108269 0.03609 0.0804479 1.82696 0.042176564 100 7016 9726 7552 0.09354 0 0.024289 0.039276 0.044358 2.787683 0.015663017  33.33333 7622 8866 8676 0.015245 0 0 0.005082 0.0050818 3.782601 0.001341671  11.11111 7740 7740 7740 0 0 0 0 0 4.782601 0 Control Cell 7740 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 11048 11856 11902 0.04775 0 0 0.015917 0.015917 0.984083 0.015916796 300 13454 12896 11798 0 0 0 0 0 1.984083 0 100 12846 13160 12546 0 0 0 0 0 2.984083 0  33.33333 12680 12458 12360 0 0 0 0 0 3.984083 0  11.11111 11602 11602 11602 0 0 0 0 0 4.984083 0 Control Cell 11602 Blank 0 Dilution 3 IC50 FALSE HBeAg: Average IC50: 0 SD: 0 HBsAg: Average IC50: 0 SD: 0

Example 5 Preparation of rSIFN-co

Preparation of lyophilized injection Lyophilized powder Stock Solution of 34.5 μg/ml rSIFN-co PB (pH 7.0) 10 mmol/L Glycine 0.4 mol/L

Preparation technique: Weigh materials according to recipe. Dissolve with sterile and pyrogen-free water. Filter through 0.22 μm membrane to de-bacterialize, preserve at 6-10° C. Fill in vials after affirming it is sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilize in freeze dryer.

Preparation of liquid injection Solution Stock Solution of 34.5 μg/ml rSIFN-co PB (pH 7.0) 25 mmol/L NaCl 0.1 mol/L

Preparation: Weigh materials according to recipe. Add to desired level with sterile and pyrogen-free water. Filter through 0.22 μm membrane to de-bacterialize, preserve at 6-10° C. Fill in airtight vial after affirming it is sterile and non-pyrogen at 0.3 ml/vial or 0.5 ml/vial. Storage at 2-10° C., and protect from light.

Example 6 Acute Toxicity of rSIFN-co

Treat mice with large dose (150 μg/kg, equal to 1000 times of the normal dose per kilo used in treatment of adult patients) of rSIFN-co at one time by intramuscular injection. Then, observe and record their deaths and toxic reactions. Results show that: 24 hours after injection, no abnormal reaction had been recorded. The organs of the animals which had been selected to be killed also had no signs of abnormal changes. Those remaining mice were all kept alive and were normal after two weeks. The weights of mice in the experimental group and control group all increased, and the ratio of increase had no obvious difference between the two groups (P>0.05) according to their weights on the fourteenth day. No abnormal changes were seen from the main organs of those mice after two weeks.

1. Experimental Material

1.1 Animals

40 healthy adult mice, weighing 18-22 g, half male and half female, qualified by Sichuan experiment animal control center.

1.2 Medicines

rSIFN-co (Provided by Sichuan Huiyang Life-engineering Ltd.) sterilized solution, 0.15 mg/ml, Lot: 981201 rSIFN-co was administered i.m. in saline.

2. Method

Separate the 40 mice into two groups randomly, one for experimental medicine, another for control. Inject medicines or saline at the same ratio (0.1 ml/10 g) through muscle to each mouse according to which group they belong. (150 μg/kg of rSIFN-co for experimental group; and saline for control group). After injection, observe and record acute toxicity shown in mice. Kill half of the mice (male and female each half) to check whether there were any abnormal pathologic changes in their main organs, such as heart, spleen, liver, lung, kidney, adrenal gland, stomach, duodenum, etc. after 24 hours. Those that remain are kept and observed until the fourteenth day. Weigh all mice, kill them, and then observe the appearance of the organs listed above to see if there are any abnormalities. Take pathological tissue and examine it, using the examination to assess the difference in weight increases in the two groups.

3. Results

Results show that there was no acute toxicity seen after all mice were treated with i.m. rSIFN-co with 150 μg/kg at a time, equal to 1000 times the normal dose per kilo used in treatment of adult patients. In the 14 days after injection, all mice lived well. They ate, drank, exercised, and excreted normally and showed normal hair conditions. None of them died. The observation of the main organs of the randomly selected mice shows no abnormal changes 24 hours after injection. 14 days after injection, all remaining mice were killed. Autopsies also showed no changes. The weights of mice in the two groups all increased, but no obvious difference was shown when accessed with statistic method (p>0.05). See Table 6.1:

TABLE 6.1 Influence to weights of mice after injection of rSIFN-co Weights Weights Increased before after value of injection injection weights Group Dose Animal (g) (g) (g) Control 0 20 19.8 ± 1.7 30.8 ± 2.8 11.0 ± 2.9 rSIFN-co 150 20 19.4 ± 1.7 32.1 ± 3.3 12.7 ± 4.3

4. Conclusion

Under conditions of this experiment, there were no toxic reactions in all mice after injection of rSIFN-co with 150 μg/kg. The conclusion can be reached that the maximum tolerable dose of i.m. in mice is 150 μg/kg, which is equal to 1000 times the normal dose per kilo used in treatment of adult patients.

Example 7 The Clinic Effects of Recombinant Super-compound Interferon (rSIFN-co)

The recombinant super-compound interferon (rSIFN-co) is an invention for viral disease therapy, especially for hepatitis. Meanwhile, it can inhibit the activity of EB viruses, VSV, Herpes simplex viruses, cornaviruses, measles viruses et al. Using Wish cells/VSV system as the assay for anti-virus activity, the results showed that: the other rIFN, was 0.9×10⁸ IU/mg, Intron A was 2.0×10⁸ IU/mg and rSIFN-co was 9×10⁸ IU/mg. The anti-viral activity of rSIFN-co is much higher than those of the former two.

Under the permission of the State Food and Drug Administration (SFDA), People's Republic of China, the clinical trials have taken place in West China Hospital, Sichuan University, the Second Hospital of Chongqing Medical University, the First Hospital of School of Medical, Zhejiang University since the February 2003. The clinical treatment which focuses on hepatitis B is conducted under the guidance of the multicenter, double-blind random test. IFN-α1b was used as control, and the primary results showed the following:

The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of Chronic Active Hepatitis B

1. Standard of patients selection: Standards 1-4 are effective for both treatment with rSIFN-co (9 μg) and IFN-α1b (5 MU, 50 μg), and Standards 1-5 are for rSIFN-co (15 μg) treatment.

1). Age: 18-65

2). HBsAg test positive over last six months, HBeAg test positive, PCR assay, HBV-DNA copies ≧10⁵/ml

3). ALT≧two times the normal value

4). Never received IFN treatment; or received the Lamividine treatment but failed or relapsed

5). Once received other IFNs (3 MU or 5 MU) treatment six months ago following the standard of SFDA, but failed or relapsed

2. Evaluation of the Effects:

In reference to the recommendations from the Tenth China National Committee of Virus Hepatitis and Hepatopathy, the effects were divided into three degrees according to the ALT level, HBV-DNA and HBeAg tests.

-   Response: ALT normal level, HBV-DNA negative, HBeAg negative -   Partial response: ALT normal level, HBV-DNA or HBeAg negative -   Non response: ALT, HBV-DNA and HBeAg unchanged -   The response and partial response groups were considered effective     cases.

3. Results of Clinic Trial:

HBsAg HBeAg HBV-DNA Transfer Transfer Transfer Heptal to to to function Effective negative negative negative Recover Period group Medicine cases Rate rate rate rate rate  8-12 A rSIFN- 32 46.88 9.38 28.12 37.50 84.38 week co(9 μg) (15) (3) (9) (12)  (2) B IFN-α1b 32 21.88 0.00  9.38 15.62 56.25 (5 MU, 50 μg)  (7) (0) (3)  (5) (18) 16-24 A rSIFN- 64 54.69 7.81 25.00 34.38 90.62 week co(9 μg) (35) (5) (16)  (22) (58) B IFN-α1b 64 25.00 0.00  9.38 18.75 78.13 (5 MU, 50 μg) (16) (0) (6) (12) (50) Group A: treatment with rSIFN-co (9 μg) Group B: treatment with IFN-α1b (5 MU, 50 μg)

In Group C, the cases were chronic active hepatitis B treatment with other IFNs (3 MU or 5 MU) before but failed or relapsed and treated with rSIFN-co (15 μg), subcutaneous injection, every one day, last 24 weeks. The total cases are 13. After 12 weeks treatment, 7 of 13 (53.85%) were effective. 3 of 13 (23.08%) HBeAg transferred to negative; 7 of 13(53.85%) HBV-DNA transferred to negative; 11 of 13 (84.62%) hepal functions recovered to normal.

4. The Side Effects of rSIFN-co Compared with IFN-α1b in the Treatment

The side effects of IFN include fever, nausea, myalgia, anorexia, hair loss, leucopenia and thrombocytopenia, etc. The maximum dose of IFN-α1b is 5 MIU per time; the routine dose is 3 MIU. When taken the routine dose, 90% patients have I-II degree (WHO standard) side effects. They are fever lower than 38° C., nausea, myalgia, anorexia, etc. When taken at maximum dose, the rate of side effects do not rise obviously, but are more serious. The maximum dose of rSIFN-co is 24 μg, subcutaneous injection, every one day for 3 months. The routine dose is 9 μg. When routine doses were used, less than 50% patients have I-II degree (WHO standard) side effects, including fever below 38° C., nausea, myalgia, anorexia, leucopenia and thrombocytopenia slightly. With maximum dosage, about 50% patients suffered from leucopenia and thrombocytopenia after using rSIFN-co one month, but those side effects would disappear after stopping treatment for one week. It is safe for continued use.

Observations of rSIFN-co Treatment of Hepatitis C

1. Standard of Patient Selection

-   -   1. Age: 18-65     -   2. HCV antibody positive     -   3. ALT≧1.5 times of the normal value, last more than 6 months

2. Evaluation of the Effects:

Referring to the standard of Infergen® for treatment of hepatitis C and according to the ALT level and HCV-RNA test, divided the effects into three degree:

Response: ALT normal level, HCV-RNA negative

Partial response: ALT normal level, HCV-RNA unchanged

Non response: ALT and HCV-RNA unchanged.

3. Effects in Clinic

The clinical trial was done at the same time with hepatitis B treatment. 46 cases received the treatment, 9 μg each time, subcutaneous injection, every day for 24 weeks. After treatment, 26 of 46 (56.52%) have obvious effects, 12 of 46 (26.08%) HCV-RNA transferred to negative, 26 of 46 (56.52%) hepal functions recovered to normal.

Example 8 Comparison of Inhibitory Effects of Different Interferons on HBV Gene Expression

Hepatitis B virus (HBV) DNA contains consensus elements for transactivating proteins whose binding activity is regulated by interferons. Treatment of HBV-infected hepatocytes with interferons leads to inhibition of HBV gene expression. The aim of the present study was to characterize the effects of different interferons on HBV regulated transcription. Using transient transfection of human hepatoma cells with reporter plasmids containing the firefly luciferase gene under the control of HBV-Enhancer (EnH) I, Enh II and core promoter, Applicant studied the biological activities of three different interferons on transcription.

Materials and Methods

1. Interferons: IFN-con1 (Infergen®), IFN-Hui-Yang (γSIFN-co) and IFN-beta 1b

2. Reporter plasmid: The DNA fragments containing HBV-Enhancer (EnH) I, Enh II and core promoter were prepared using PCR and blunt-end cloned into the Smal I site of the promoter- and enhancer-less firefly luciferase reporter plasmid pGL3-Basic (Promega, Wis., USA). The resulting reporter plasmid was named as pGL3-HBV-Luc.

3. Cell Culture and DNA transfection: HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin and 100 ug/ml streptomycin. The cells were kept in 30° C., 5% CO2 incubator. The cells were transfected with pGL3-HBV-Luc reporter plasmid using Boehringer's Lipofectin transfection kit. After 18 hours, the medium containing transfection reagents was removed and fresh medium was added with or without interferons. The cells were kept in culture for another 48 hours.

4. Luciferase Assay: Forty-eight hours after addition of interferon, the cells were harvested and cell lysis were prepared. The protein concentration of cell lysates were measured using Bio-Rad Protein Assay kit. The luciferase activity was measured using Promega's Luciferase Reporter Assay Systems according to the instructions of manufacturer.

Results

Expression of Luciferase Activity in Different Interferon—Treated Cell Lysates

No treatment IFN-con1 IFN-Hui-Yang IFN-beta 1b 100 48 + 8 29 + 6 64 + 10

This result shows that γSIFN-co inhibits most effectively on the expression of HBV gene expression.

Example 9 Side Effects and Changes in Body Temperature when Using γSIFN-co

There are usually more side effects to using interferon. The side effects includes: nausea, muscle soreness, loss of appetite, hair loss, hypoleucocytosis (hypoleukmia; hypoleukocytosis; hypoleukia), and decrease in blood platelets, etc.

Method

Sample patients are divided into two groups. 11 patients in Group A were injected with 9 μg Infergen®. 10 patients in Group B were injected with 9 μg γSIFN-co. Both groups were monitored for 48 hours after injections. First monitoring was recorded 1 hour after injection, after that, records were taken every 2 hours.

Table 9.1 is the comparison of side effects between patients being injected with 9 μg of Infergen® and 9 μg of γSIFN-co.

TABLE 9.1 Side Effects γSIFN-co Infergen ® 9 μg 9 μg Person: n = 10 Person: n = 11 Body Systems Reactions Headcount Headcount In General Feebleness 3 3 Sole heat 1 frigolability 3 4 Lack of 3 strength in legs Mild lumbago 2 1 Body soreness 4 5 Central Nervous Headache 3 6 System/ Dizziness 2 11 Peripheral Drowsiness 3 Nervous System Gastroenterostomy Apoclesis 1 Celiodynia 1 Diarrhea 1 Musculoskeletal Myalgia 1 2 system Arthralgia 2 Respiratory Stuffy nose 1 system Paropsia Swollen Eyes 1

Result

For those patients who were injected with γSIFN-co, the side effects were minor. They had some common symptoms similar to flu, such as: headache, feebleness, frigolability, muscle soreness, hidrosis, arthralgia (arthrodynia; arthronalgia). The side effects of those patients whom were injected with Infergen® were worse than those injected with γSIFN-co.

From FIGS. 8A, 8B, 8C, and 8D, it was obvious that the body temperatures of sample patients in Group A were higher than the patients in Group B. It also reflected that the endurance of γSIFN-co was much better than Infergen®.

Example 10 Crystal Growth of γSIFN-co and Test of Crystallography Parameter

Crystal γSIFN-co. Two types of crystal were found after systematically trial and experiment. (See FIGS. 9-11)

1. Crystal Growth

-   -   Dissolve γSIFN-co protein with pure water (H2O) to 3 mg/ml in         density. Search of crystallization by using Hampton Research         Crystal Screen I and II which was made by Hampton Company. By         using Drop Suspension Diffusion Method, liquid 500 μl, drop 1 μl         protein+1 μl liquid, in 293K temperature. First 2 different         types of small crystals were found as listed in Table 10.1.

TABLE 10.1 Screen of γSIFN-co Crystallin Condition I II Diluent 0.1M Tris-HCl 0.1M HEPES PH = 8.75 PH = 7.13 Precipitant 17.5% (w/v) PEG550 MME 10% (w/v)PEG6K Additives 0.1M NaCl 3% (v/v)MPD Temperature 293 K 293 K Crystal Size (mm) 0.2 × 0.2 × 0.1 0.6 × 0.02 × 0.02 Crystallogram FIG. 9 FIG. 10

2. Data Collection and Processing

-   -   Crystal I was used to collect X-Ray diffraction data and         preliminary analysis of crystallography. Parameters were also         tested. The diffraction data was collected under the room         temperature. The Crystal I (Condition I) was inserted into a         thin siliconized wall tube. Using BrukerAXS Smart CCD detector,         the light source is CuKα (λ=1.5418 Å) generated by Nonius FR591         X-ray generator. Light power 2000 KW (40 kv×50 mA), wave length         1.00 Å, under explosion 60 second, Δσ=2°, the distance between         crystal and detector was 50 mm. Data was processed using Proteum         Procedure Package by Bruker Company. See FIG. 11 for crystal         diffraction pattern (partially). See Table 10.2 for the result         of the process.

TABLE 10.2 Results of Crystallography Parameters Parameters a (Å) 82.67 b (Å) 108.04 c (Å) 135.01 α (°) 90.00 β (°) 90.00 γ (°) 98.35 Space Group P2 or P2₁ Sharpness of separation 5 Å Asymmetric molecule # 10 Dissolution 57.6%

In addition, there was no crystal growth of γSIFN-co based on previous publications. The closest result to the γSIFN-co was huIFN-a2b but the screen was very complicated. After seeding 3 times, crystal grew to 0.5×0.5×0.3 mm, sharpness of separation was 2.9 Å, space group was P2₁. The crystals were also big, asymmetric molecule number was 6, and dissolution was about 60%. 

1. A method for treating viral disease in a subject comprising administering to the subject an effective amount of a recombinant interferon encoded by a polynucleotide having the sequence of SEQ ID NO:1, wherein the recombinant interferon has the amino acid sequence of SEQ ID NO:2, and the recombinant interferon has increased inhibitory activities on the expression of Hepatitis B surface antigen (HbsAg) and Hepatitis B e antigen (HbeAg) as compared to an interferon not encoded by SEQ ID NO:1.
 2. The method of claim 1, wherein the viral disease is caused by hepatitis A virus, hepatitis B virus, hepatitis C virus, other types of hepatitis virus, Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, other types of herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia viruses I, human T-cell leukemia viruses II, or human T-cell leukemia virus III.
 3. The method of claim 1, wherein the interferon is administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via an inspirator.
 4. The method of claim 1, wherein the interferon is administered following the protocol of 9 μg or 15 μg per injection, 3 times a week, for a total of 24 weeks.
 5. A method for treating tumors in a subject comprising administering to the subject an effective amount of a recombinant interferon encoded by a polynucleotide having the sequence of SEQ ID NO:1, wherein the recombinant interferon has the amino acid sequence of SEQ ID NO:2, and the recombinant interferon has increased inhibitory activities on the expression of HBsAg and HBeAg of Hepatitis B Virus as compared to an interferon not encoded by SEQ ID NO:1.
 6. The method of claim 5, wherein the interferon is administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via an inspirator.
 7. The method of claim 5, wherein the interferon is administered following the protocol of 9 μg or 15 μg per injection, 3 times a week, for a total of 24 weeks. 